Conjugates are widely used in bioscience research, diagnostics and medicine. In the simplest case conjugates take the form of a first chemical entity (A), typically a molecule such as a biomolecule, that is linked to a second chemical entity (B), such as a label molecule, to form an AB hybrid. Oligomeric forms, represented by the formula AjBk, where j and k are integers, are also possible. Conjugates are usually designed for a specific purpose and often involve novel combinations of materials that are not naturally occurring. Typically, one component of the conjugate has the capacity to interact with other molecules (e.g. antigens), e.g. being an antibody, and the second component adds some other useful property (e.g. measurability, ability to kill cancer cells), e.g. being a label.
Conjugates of the present invention may comprise combinations of entities, where A and/or B may comprise one of the following: antibodies, antibody fragments, nucleic acids, beads, polymers, liposomes, carbohydrates, fluorescent proteins and dyes, peptides, radionuclides, toxins, gold particles, streptavidin, biotin, enzymes, chelating agents, haptens, drugs and many other molecules. This list encompasses a vast array of molecules and thus the number of possible combinations in conjugates is almost limitless. It follows that there is considerable scope to create novel hybrid molecules with unusual or unique properties.
One of the most important applications of immunoconjugates is in the quantitation and/or detection of antigens, which are often presented on a surface. For example, in western blotting applications the antigen is immobilised on a sheet of nitrocellulose; in an enzyme-linked immunadsorbent assay (ELISA), the antigen is adsorbed on surface of a polystyrene plate; in immunohistochemistry, the antigen is embedded, along with many other proteins, in a thin slice of tissue, which is attached to a glass slide. While these techniques differ fundamentally in the way in which the antigen is presented to the conjugate, the choice of detection methods is essentially the same. There are two main types. With direct detection, the ‘primary’ antibody (i.e. the antibody that binds to the antigen) is conjugated to a label that can be measured with a suitable measuring device. With indirect detection, the label is introduced via a secondary reagent, which binds to the primary antibody. The secondary reagent most often is an antibody conjugate comprising a secondary antibody conjugated to a label. More complex detection strategies exist but each of these generally is a variation on one of the above two themes.
With indirect methods, one secondary reagent can be used with a range of unlabelled primary antibodies, which is extremely convenient, although the need for more incubation and wash steps than with direct methods is a major disadvantage. There is also potential for unwanted cross-reactivity of the secondary antibody with immobilised antigens. While direct detection methods offer considerable advantages in terms of speed, cost, and data quality, indirect methods currently predominate. The explanation for this fact is that most primary antibodies are available commercially only in an unlabeled form. Moreover, these reagents are expensive and usually cannot be purchased by researchers in quantities that allow cost-effective production of labelled conjugates using current labelling methodologies.
In order to produce a conjugate, a bifunctional reagent that contains two reactive groups is generally used to link the two components of interest. The reactive groups on the bifunctional reagent are either identical in functionality (‘homobifunctional’) or different in functionality (‘heterobifunctional’). The best-known example of a homobifunctional reagent is the bis-aldehyde glutaraldehyde, which reacts with amines (or hydrazides). Since most biomolecules contain multiple amines, the use of glutaraldehyde commonly results in the formation of high molecular weight conjugates. Furthermore, the polymeric nature of solutions of glutaraldehyde, which can vary considerably with age, means that conjugates prepared with glutaraldehyde are generally quite difficult to reproduce.
Heterobifunctional reagents are generally preferred in the preparation of conjugates as they allow the operator to exert a higher degree of control over the conjugation process. A popular heterobifunctional conjugation strategy involves the coupling of an amine group on one molecule (B) to a free sulfhydryl group (SH) on another molecule (A) via a heterobifunctional reagent (X-Y) having an amine-reactive moiety (X) and a sulfhydryl-reactive moiety (Y). A ‘spacer’ often separates the reactive moieties of the heterobifunctional reagent; there are many heterobifunctional reagents that have varying spacer structures but which share essentially the same chemical reactivity.
Typically, one biomolecule (B) to be conjugated is reacted via its amine groups with the X functionality of the heterobifunctional reagent, resulting in a B-Y derivative. Excess heterobifunctional reagent is then removed and purified B-Y is reacted with sulfhydryl groups on the other molecule (A). X is commonly an N-hydroxysuccinimide (NHS) ester, while Y may be one of several moieties. Y may or may not be integrated into the final AB conjugate. The sulfhydryl group derived from A is almost always incorporated either as a stable thioether bond or as one half of a reversible (reducible) disulphide bridge between A and B. Y may be any sulfhydryl-reactive functionality including: maleimide, epoxide, iodoacetyl, bromoacetyl, pyridyldithiol, methanethiosulfonate, and the like.
Examples of amine and sulfhydryl reactive heterobifunctional reagents include: N-succinimidyl 3-(2 pyridyldithio) propionate (SPDP); variants of SPDP with extended spacers (LC-SPDP; LC=‘long chain’) and sulfo groups to increase aqueous solubility (sulfo-LC-SPDP); succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT); sulfo-LC-SMPT; Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC); sulfo-SMCC; m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS); sulfo-MBS; N-Succinimidyl(4-iodoacetyl)aminobenzoate (SIAB); sulfo-SIAB; Succinimidyl-4-(p-maleimidophenyl)butyrate (SMBP); sulfo-SMBP; N-(γ-Maleimidobutyryloxy)succinimide ester (GMBS); sulfo-GMBS; Succinimidyl-6-((iodoacetyl)amino)hexanoate (SIAX); and its extended spacer form SIAXX; Succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (SIAC); and its extended spacer form (SIACX); p-Nitrophenyl iodoacetate (NPIA). There are many other related examples, such as the carbonyl and sulfhydryl-reactive linker, β-maleimidopropionic acid hydrazide (BMPH).
The sulfhydryl groups on A may be indigenous. However, more commonly sulfhydryl groups are not present and need to be introduced by a thiolation reaction prior to the conjugation step. In the case of antibodies, thiol groups may be generated by means of a reducing agent (e.g. MEA or dithiothreitol (DTT)), which break disulfide bridges at various positions on the antibody molecule. Alternatively, techniques are known by which other functional groups (commonly amines) can be modified to introduce either a free sulfhydryl group or a protected sulfhydryl group, which can then be deprotected by treatment with a reducing agent to generate a thiolated product (i.e. A-SH). In the known conventional techniques, prior to conjugation with B-Y at least one separation step is required to separate the desired thiolated product A-SH from unreacted thiolation reagent, and any by-products including free sulfhydryl groups that would compete for conjugation to B-Y and possibly also reducing agent that would otherwise compete with A-SH for conjugation to B-Y. Separation is performed by techniques including desalting on chromatography columns, gel filtration, dialysis, or washing. The separation step or steps inevitably result in losses and dilution of material. Because of the tedious nature of the separating step(s) and/or requirement for significant quantities of A, the thiolation step may never be thoroughly optimised.
By way of example, 2-iminothiolane (2IT), which is also known as Traut's reagent (Traut et al., Biochemistry 12, 3266-3273, 1973) has previously been used to introduce SH groups into proteins, particularly antibodies. The reagent reacts with primary amines (e.g. present on lysine) and generates a terminal sulfhydryl group in a ring-opening reaction. Excess Traut's reagent is removed, typically by desalting, prior to conjugation of the resulting thiolated molecule with a thiol-reactive group on another molecule. Although not mentioned in otherwise comprehensive works on bioconjugation chemistry (e.g. Bioconjugate Techniques; G. T Hermanson, Academic Press 1996), 2IT also undergoes a secondary reaction in which the nascent thiol reacts intramolecularly to form an unreactive thioester (Bartlett & Busch., Biol. Mass Spectrom. 23, 353-356, 1994; Singh et al. Anal. Biochem. 236, 114-125, 1996). It is clear from the known chemistry of Traut's reagent that the duration of the thiolation reaction may be critical to the success of the subsequent conjugation step, and that desalting or other separation steps need to be completed quickly.
Conventional prior uses of 2IT in the production of conjugates with molecules engineered to contain thiol-reactive functions employ excess 2IT followed by a desalting, dialysis or wash step. This type of approach is recommended by suppliers of 2IT (e.g. Pierce technical bulletin 0414; product 26101) and of products used in the preparation of bioconjugates (e.g. Prozyme TechNote #TNPJ300). Other publications that describe this approach include: U.S. Pat. Nos. 6,962,703, 6,936,701, 6,669,938, 6,010,902, 5,869,045, 5,164,311; Stanisic et al., Infection and Immunity 71, 5700-5713, 2003; Mandler et al., Journal of the National Cancer Institute, 92, 1573-1581, 2000; Huwyler et al., Proc Natl Acad Sci 93, 14164-14169, 1996.
One potentially promising solution to the problem of desalting was suggested (Haughland. Handbook of Fluorescent Probes and Research Chemicals, 6th edition, Molecular Probes, p 49) which involved reduction of protected sulfhydryl groups by TCEP (Tris(2-carboxyethyl)phosphine). While it is claimed that removal of TCEP is unnecessary, as it does not interfere with subsequent conjugation steps, Getz et al (Anal Biochem 273, 73-80, 1999) showed significant interference of TCEP in conjugation reactions. Moreover, Shafer et al (Anal Biochem 282, 161-164, 2000) reported that TCEP combines rapidly with the sulfhydryl-reactive maleimide and iodoacetyl groups. Furthermore, bioconjugation reactions commonly are carried out in phosphate buffers at pH 7-8, under which conditions TCEP is unstable (Han & Han, Anal Biochem 220, 5-10, 1994). TCEP is very stable at extremes of pH (e.g. in 10 mM HCl or in 100 mM NaOH), which are not compatible with most biomolecules. While TCEP has found certain niche applications its serious limitations have ensured that the preferred methods for producing bioconjugates have changed little since it became commercially available in 1992. TCEP does not contain a sulphur atom and therefore, for present purposes, is not considered a “thiol generator”.
McCall et al (1990 Bioconjugate Chem. 1, 222-226) disclosed a one step method for conjugating macrocyclic chelators to antibodies using 2IT. Specifically they used 2IT to join 6-[p(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-N,N1,N11,N111-tetracetic acid, abbreviated as BAT, or a similar compound, 2-[p(bromoacetamido)benzyl]-1,4,7,10-tetraazacyclododecane-N,N1N11,N111-tetra acetic acid (abbreviated as BAD), to a mouse antibody. The BAT/BAD reagents were monovalent with respect to sulfhydryl reactive groups i.e. having only one group per molecule able to react with a sulfhydryl group. McCall et al suggested that the “one step” method disclosed therein was applicable only to the particular BAT/BAD reagent (“since under mildly alkaline conditions bromoacetamide reagents react rapidly with sulfhydryl groups but only slowly with amino groups, the antibody, BAT and 2 IT solutions could be combined in a single reaction mixture”). There is no suggestion that this technique might be generally applicable and the standard method used commercially remains a 2 step approach with an intervening desalting, purification or washing stage.